Cell wall and enzyme changes during the graviresponse of the leaf-sheath pulvinus of oat (Avena sativa)

The graviresponse of the leaf-sheath pulvinus of oat (Avena sativa) involves an asymmetric growth response accompanied by several asymmetric processes, including degradation of starch and cell wall synthesis. To understand further the cellular and biochemical events associated with the graviresponse, changes in cell walls and their constituents and the activities of related enzymes were investigated in excised pulvini. Asymmetric increases in dry weight with relatively symmetric increases in wall weight accompanied the graviresponse. Starch degradation could not account for increases in wall weight. However, a strong asymmetry in invertase activity indicated that hydrolysis of exogenous sucrose could contribute significantly to the increases in wall and dry weights. Most cell wall components increased proportionately during the graviresponse. However, beta-D-glucan did not increase symmetrically, but rather increased in proportion in lower halves of gravistimulated pulvini. This change resulted from an increase in glucan synthase activity in lower halves. The asymmetry of beta-D-glucan content arose too slowly to account for initiation of the graviresponse. A similar pattern in change in wall extensibility was also observed. Since beta-D-glucan was the only wall component to change, it is hypothesized that this change is the basis for the change in wall extensibility. Since wall extensibility changed too slowly to account for growth initiation, it is postulated that asymmetric changes in osmotic solutes act as the driving factor for growth promotion in the graviresponse, while wall extensibility acts as a limiting factor during growth.     

Changes in the ultrastructure of cell walls, cellulose synthesis, and glucan synthase activity from gravistimulated pulvini of oat (Avena sativa)

Ultrastructural analyses of the cell walls from top and bottom halves of gravistimulated pulvini from oat leaves show a decrease in the density of material within the cell walls from the lower halves of pulvini after 24 h of gravistimulation. Assays of cellulose synthesis with a 14C-sucrose pulse-chase experiment indicate no difference in the amount of new cellulose synthesized in top compared with bottom halves of gravistimulated pulvini. The highest rate of cellulose synthesis occurs with 12-24 h of gravistimulation. Treatment of graviresponding pulvini with 2,6-dichlorobenzonitrile (DCBN) had only a minor effect on segment gravitropic curvature. We also found that there is no difference in the activities of either glucan synthase I or glucan synthase II in top halves as compared with bottom halves of gravistimulated pulvini. We conclude that the graviresponse in oat stems is not driven by new cell wall synthesis but, rather, by changes in cell wall plasticity and osmotic potential.     

The role of calcium in growth induced by indole-3-acetic acid and gravity in the leaf-sheath pulvinus of oat (Avena sativa)

Leaf-sheath pulvini of excised segments from oat (Avena sativa L.) were induced to grow by treatment with 10 M indole-3-acetic acid (IAA), gravistimulation, or both, and the effects of calcium, EGTA, and calcium channel blockers on growth were evaluated. Unilaterally applied calcium (10 mM CaCl₂) significantly inhibited IAA-induced growth in upright pulvini but had no effect on growth induced by either gravity or gravity plus IAA. Calcium alone had no effect on upright pulvini. The calcium chelator EGTA alone (10 mM) stimulated growth in upright pulvini. However, EGTA had no effect on either IAA-or gravity-induced growth but slightly diminished growth in IAA-treated gravistimulated pulvini. The calcium channel blockers lanthanum chloride (25 mM), verapamil (2.5 mM), and nifedipine (2.5 mM) greatly inhibited growth as induced by IAA (50% inhibition) or IAA plus gravity (20% inhibition) but had no effect on gravistimulated pulvini. Combinations of channel blockers were similar in effect on IAA action as individual blockers. Since neither calcium ions nor EGTA significantly affected the graviresponse of pulvini, we conclude that apoplastic calcium is unimportant in leaf-sheath pulvinus gravitropism. The observation that calcium ions and calcium channel blockers inhibit IAA-induced growth, but have no effect on gravistimulated pulvini, further supports previous observations that gravistimulation alters the responsiveness of pulvini to IAA.     

How cereal grass shoots perceive and respond to gravity

The leaf-sheath pulvinus of grasses presents a unique system for studying gravitropism, primarily because of its differences from other organs. The mature pulvinus is a discrete organ specialized for gravitropism: it is nongrowing in the absence of gravistimulation and capable of displaying a graviresponse independent of the rest of the plant. In this paper we present a model for gravitropism in pulvini based on recent findings from studies on the mechanisms of graviperception and graviresponse. According to this model, amyloplasts play an essential role in perceiving a change in the orientation of the pulvinus. The perception of this reorientation leads to the enhanced synthesis and release from conjugate of the auxin IAA, and the increased conjugation of gibberellin, on a localized basis. Because there is a graded growth promotion across the gravistimulated pulvinus, it is suggested that the observed hormonal asymmetry is actually an indication of a linear gradient of hormone concentration, as well as hormone response, across the pulvinus. It is further suggested that the linear gradient of hormone concentration may be predominantly the result of local changes in hormone level, rather than a product of hormonal movement into or across the pulvinus.     

Altered Growth Response to Exogenous Auxin and Gibberellic Acid by Gravistimulation in Pulvini of Avena sativa

Pulvini of excised segments from oats (Avena sativa L. cv Victory) were treated unilaterally with indoleacetic acid (IAA) or gibberellic acid (GA₃) with or without gravistimulation to assess the effect of gravistimulation on hormone action. Optimum pulvinus elongation growth (millimeters) and segment curvature (degrees) over 24 hours were produced by 100 micromolar IAA in vertical segments. The curvature response to IAA at levels greater than 100 micromolar, applied to the lower sides of gravistimulated (90°) pulvini, was significantly less than the response to identical levels in vertical segments. Furthermore, the bending response of pulvini to 100 micromolar IAA did not vary significantly over a range of presentation angles between 0 and 90°. In contrast, the response to IAA at levels less than 10 micromolar, with gravistimulation, was approximately the sum of the responses to gravistimulation alone and to IAA without gravistimulation. This was observed over a range of presentation angles. Also, GA₃ (0.3-30 micromolar) applied to the lower sides of horizontal segments significantly enhanced pulvinus growth and segment curvature, although exogenous GA₃ over a range of concentrations had no effect on pulvinus elongation growth or segment curvature in vertical segments. The response to GA₃ (10 micromolar) plus IAA (1.0 or 100 micromolar) was additive for either vertical or horizontal segments. These results indicate that gravistimulation produces changes in pulvinus responsiveness to both IAA and GA₃ and that the changes are unique for each growth regulator. It is suggested that the changes in responsiveness may result from processes at the cellular level other than changes in hormonal sensitivity.     

Competency for graviresponse in the leaf-sheath pulvinus of Avena sativa: onset to loss

The development of the leaf-sheath pulvinus of oat (Avena sativa L. cv. Victory) was studied in terms of its competency to respond to gravistimulation. Stages of onset of competency, maximum competency and loss of competency were identified, using the length of the supertending internode as a developmental marker. During the early phases in the onset of competency, the latency period between stimulus and graviresponse decreased and the steady state response rate increased significantly. When fully competent, the latency period remained constant as the plant continued to develop, suggesting that the latency period is relatively insensitive to quantitative changes (e.g., in carbohydrate or nutrient availability) at the cell level within the plant. In contrast, the response rate was found to increase with plant development, indicating that graviresponse rate is more strongly influenced by quantitative cellular changes. The total possible graviresponse of a single oat pulvinus was confirmed to be significantly less than the original presentation angle. This was shown to not result from a loss of competency, since the graviresponse could be reinitiated by increasing the presentation angle. As a result of the low overall graviresponse of individual pulvini, two or more pulvini are required to bring the plant apex to the vertical. This was determined to occur though the sequential, rather than simultaneous, action of successive pulvini, since a given pulvinus lost competency to gravirespond shortly after the next pulvinus became fully competent.     

Elemental analysis of freeze-dried thin sections of Samanea motor organs: barriers to ion diffusion through the apoplast

Leaflet movements in the legume Samanea saman are dependent upon massive redistribution of potassium (K), chloride (Cl), and other solutes between opposing (extensor and flexor) halves of the motor organ (pulvinus). Solutes are known to diffuse through the apoplast during redistribution. To test the possibility that solute diffusion might be restricted by apoplastic barriers, we analyzed elements in the apoplast in freeze-dried cryosections of pulvini using scanning electron microscopy/x-ray microanalysis. Large discontinuities in apoplastic K and Cl at the extensor-flexor interface provide evidence for a barrier to solute diffusion. The barrier extends from the epidermis on upper and lower sides of the pulvinus to cambial cells in the central vascular core. It is completed by hydrophobic regions between phloem and cambium, and between xylem rays and surrounding vascular tissue, as deduced by discontinuities in apoplastic solutes and by staining of fresh sections with lipid-soluble Sudan dyes. Thus, symplastic pathways are necessary for ion redistribution in the Samanea pulvinus during leaflet movement. In pulvini from leaflets in the closed state, all cells on the flexor side of the barrier have high internal as well as external K and Cl, whereas cells on the extensor side have barely detectable internal or external K or Cl. Approximately 60% of these ions are known to migrate to the extensor during opening; all return to the flexor during subsequent closure. We propose that solutes lost from shrinking cells in the outer cortex diffuse through the apoplast to plasmodesmata-rich cells of the inner cortex, collenchyma, and phloem; and that solutes cross the barrier by moving through plasmodesmata.     

Changes in mitogen-activated protein kinase activity occur in the maize pulvinus in response to gravistimulation and are important for the bending response

The maize (Zea mays L.) pulvinus was used as a model system to study the signalling events that lead to differential growth in response to gravistimulation in plants. The pulvinus functions to return tipped plants to vertical via differential elongation of the cells on its lower side. By performing immunokinase assays using total soluble protein extracts and an antibody against mammalian ERK1, a mitogen‐activated protein kinase (MAPK)‐like activity was assayed in pulvini halves harvested at various time points after tipping. We detected a reproducible alternation of higher levels of activity occurring between the upper and lower halves of the pulvinus between 75 and 180 min after tipping, with a sustained increase in the upper half occurring at the end of the time‐course. This timing roughly corresponds to the presentation time for maize (i.e. the amount of time that the plant needs to be tipped before it is committed to bend), which occurs between 2 and 4 h. Treatment of maize stem explants with an inhibitor of MAPK activation, U0126, led to a reduction in the activity of this kinase, as well as an almost 65% reduction in bending as measured at 20 h. Rinsing out of the inhibitor resulted in recovery of both bending and kinase activity. It is possible that changes in MAPK activity in the gravistimulated pulvinus are part of a signalling cascade that may help to distinguish between minor perturbations in plant orientation and more significant and long‐term changes, and may also help to determine the direction of bending.     

Water Relations in Pulvini from Samanea saman: I. Intact Pulvini

The movement of Samanea leaflets depends upon changes in the curvature of the pulvinus at the base of each leaflet. Pulvinar bending and straightening, in turn, are driven by the movement of water between opposing (extensor and flexor) sides of the pulvinus. Although water movement depends on water potential (Ψ) and thus on osmotic potential (π) and hydrostatic pressure (P), none of these parameters have been measured in Samanea. In this investigation, Ψ and π were measured and P was calculated for extensor and flexor tissues of excised, whole pulvini that were open in the light and closed in the dark. In fully open pulvini, π in the extensor was generally between 800 and 1000 milliosmol per kilogram and exceeded π in the flexor by 300 to 450 milliosmol per kilogram. In fully closed pulvini the reverse was true, with π in the flexor between 800 and 1000 milliosmol per kilogram, exceeding π in the extensor by 300 to 450 milliosmol per kilogram. To obtain approximate values of Ψ of pulvinar tissues, shallow cuts in extensor and flexor sides of oil-covered pulvini were filled with droplets of polyethylene glycol solutions of known Ψ. Droplets maintaining constant size were assumed to have the same Ψ as the tissue. Extensor and flexor halves of open pulvini had very different Ψ (extensor, about −1.4 MPa; flexor, about −0.3 MPa), but both sides of closed pulvini had similar Ψ (about −0.3 MPa). Measurements of Ψ and π and calculations of P indicate: (a) In open pulvini, P is about the same in extensor and flexor. The large Ψ gradient is caused by a large osmotic gradient. (b) In closed pulvini, P is approximately 50% higher in the flexor than in the extensor. This difference in P compensates for differences in π such that the Ψ gradient is small. (c) Pulvini close as P increases in the flexor and reopen as flexor P decreases; extensor P values are similar in open and closed pulvini.     

Do Starch Statoliths Act as the Gravisensors in Cereal Grass Pulvini?

To determine if starch statoliths do, in fact, act as gravisensors in cereal grass shoots, starch was removed from the starch statoliths by placing 45-day-old intact barley plants (Hordeum vulgare cv `Larker') in the dark at 25°C for 5 days. Evidence from staining with I₂-KI, scanning electron microscopy, and transmission electron microscopy indicated that starch grains were no longer present in plastids in the pulvini of plants placed in the dark for 5 days. Furthermore, gravitropic curvature response in these pulvini was reduced to zero, even though pulvini from vertically oriented plants were still capable of elongating in response to applied auxin plus gibberellic acid. However, when 0.1 molar sucrose was fed to the dark pretreated, starch statolith-free pulvini during gravistimulation in the dark, they not only reformed starch grains in the starch-depleted plastids in the pulvini, but they also showed an upward bending response. Starch grain reformation appeared to precede reappearance of the graviresponse in these sucrose-fed pulvini. These results strongly support the view that starch statoliths do indeed serve as the gravisensors in cereal grass shoots.     

Gravistimulation leads to asymmetry of both auxin and gibberellin levels in barley pulvini

The auxin indole-3-acetic acid (IAA) is known to promote the biosynthesis of active gibberellins (GAs) in barley ( Hordeum vulgare ). We therefore investigated the possibility that this interaction might contribute to the gravitropic response of barley leaf sheath pulvini. Barley plants at the inflorescence stage were gravistimulated for varying times, and the pulvini were then separated into upper and lower halves for quantification of IAA and GAs by GC-MS. Consistent with the Cholodny–Went theory, the lower portion contained more IAA than did the upper portion. This difference was detected as early as 2.5 h after the start of gravistimulation, and bending was also observed at this stage. At later time points tested (6 h and 24 h), but not at 2.5 h or 3 h, the higher auxin content of the lower half was associated with a higher level of GA₁, the main bioactive GA in barley. Consistent with that result, the expression of Hv3ox2 , which encodes a key enzyme for the conversion of GA₂₀ to GA₁, was higher in the lower side than in the upper, after 6 h. It is suggested that in gravistimulated leaf sheath pulvini, auxin accumulates in the lower side, leading to a higher level of GA₁, which contributes to the bending response. Further evidence that GAs play a role in the gravitropic response was obtained from GA-related mutants, including the elongated sln1c mutant, in which GA signalling is constitutive. Pulvinar bending in the sln1c mutant was greater than in the wild-type. This result indicates that in the lower side of the gravistimulated pulvinus, the relatively high level of bioactive GA facilitates, but does not mediate, the bending response.     

A role for inositol 1,4,5-trisphosphate in gravitropic signaling and the retention of cold-perceived gravistimulation of oat shoot pulvini

Plants sense positional changes relative to the gravity vector. To date, the signaling processes by which the perception of a gravistimulus is linked to the initiation of differential growth are poorly defined. We have investigated the role of inositol 1,4,5-trisphosphate (InsP(3)) in the gravitropic response of oat (Avena sativa) shoot pulvini. Within 15 s of gravistimulation, InsP(3) levels increased 3-fold over vertical controls in upper and lower pulvinus halves and fluctuated in both pulvinus halves over the first minutes. Between 10 and 30 min of gravistimulation, InsP(3) levels in the lower pulvinus half increased 3-fold over the upper. Changes in InsP(3) were confined to the pulvinus and were not detected in internodal tissue, highlighting the importance of the pulvinus for both graviperception and response. Inhibition of phospholipase C blocked the long-term increase in InsP(3), and reduced gravitropic bending by 65%. Short-term changes in InsP(3) were unimpaired by the inhibitor. Gravitropic bending of oat plants is inhibited at 4 degrees C; however, the plants retain the information of a positional change and respond at room temperature. Both short- and long-term changes in InsP(3) were present at 4 degrees C. We propose a role for InsP(3) in the establishment of tissue polarity during the gravitropic response of oat pulvini. InsP(3) may be involved in the retention of cold-perceived gravistimulation by providing positional information in the pulvini prior to the redistribution of auxin.     

Gibberellins and Gravitropism in Maize Shoots : Endogenous Gibberellin-Like Substances and Movement and Metabolism of [3H]Gibberellin A20

[³H]Gibberellin A₂₀ (GA₂₀) of high specific radioactivity (49.9 gigabecquerel per millimole) was applied equilaterally in a ring of microdrops to the internodal pulvinus of shoots of 3-week-old gravistimulated and vertical normal maize (Zea mays L.), and to a pleiogravitropic (prostrate) maize mutant, lazy (la). All plants converted the [³H]GA₂₀ to [³H]GA₁⁻ and [³H]GA₂₉-like metabolites as well as to several metabolites with the partitioning and chromatographic behavior of glucosyl conjugates of [³H]GA₁, [³H]GA₂₉, and [³H]GA₈. The tentative identification of these putative [³H]GA glucosyl conjugates was further supported by the release of the free [³H]GA moiety after cleavage with cellulase. Within 12 hours of the [³H]GA₂₀ feed, there was a significantly higher proportion of total radioactivity in lower than in upper halves of internode and leaf sheath pulvini in gravistimulated normal maize. Further, there was a significantly higher proportion of putative free GA metabolites of [³H]GA₂₀, especially [³H]GA₁, in the lower halves of normal maize relative to upper halves. The differential localization of the metabolites between upper and lower halves was not apparent in the pleiogravitropic mutant, la. Endogenous GA-like substances were also examined in gravistimulated maize shoots. Forty-eight hours after gravistimulation of 3-week-old maize seedlings, endogenous free GA-like substances in upper and lower leaf sheath and internode pulvini halves were extracted, chromatographed, and bioassayed using the `Tanginbozu' dwarf rice microdrop assay. Lower halves contained consistently higher total levels of GA-like activity. The qualitative elution profile of GA-like substances differed consistently, upper halves containing principally a GA₂₀-like substance and lower halves containing mainly GA₁-like and GA₁₉-like substances. Gibberellins A₁ (10 nanograms per gram) and A₂₀ (5 nanograms per gram) were identified from these lower leaf sheath pulvini by capillary gas chromatography-selected ion monitoring. Results from all of these experiments are consistent with a role for GAs in the differential shoot growth that follows gravitropism, although the results do not eliminate the possibility that the redistribution of GAs results from the gravitropic response.     

Increased levels of reactive oxygen species and expression of a cytoplasmic aconitase/iron regulatory protein 1 homolog during the early response of maize pulvini to gravistimulation

The maize (Zea mays L.) stem pulvinus is a disc of tissue located apical to each node that functions to return a tipped stem to a more upright position via increased cell elongation on its lower side. We investigated the possibility that reactive oxygen species (ROS) and hydrogen peroxide (H2O2), in particular, are involved in the gravitropic response of the pulvinus prior to initiation of the growth response by employing the cytochemical stain 3,3'-diaminobenzidine (DAB). DAB polymers were found in the bundle sheath cells of gravistimulated pulvini in association with amyloplasts after 1 min of gravistimulation, and the signal spread throughout the cytosol of these cells by 30 min. Furthermore, treatment of maize stem explants containing pulvini with 1 mm H2O2 on their upper sides caused reversal of bending polarity. Similar, though less dramatic, results were obtained via application of 1 mm ascorbic acid to the lower side of the explants. In addition, we determined that a maize cytoplasmic aconitase/iron regulatory protein 1 (IRP1) homolog is up-regulated in the pulvinus bundle sheath cells after gravistimulation using suppressive subtractive hybridization PCR (SSH PCR), real-time RT-PCR and in situ hybridization. Although we do not yet know the role of the IRP1 homolog in the pulvinus, the protein is known to be a redox sensor in other systems. Collectively, our results point to an increase in ROS quite early in the gravitropic signalling pathway and its possible role in determining the direction of bending of the pulvini. We speculate that an ROS burst may serve to link the physical phenomenon of amyloplast sedimentation to the changes in cellular biochemistry and gene expression that facilitate directional growth.     

Effects of staurosporine, okadaic acid and sodium fluoride on protein phosphorylation in graviresponding oat shoot pulvini.

This study focuses on the characterization of protein phosphorylation in the gravitropic response in oat shoot pulvini through the use of inhibitors of this process, namely staurosporine, okadaic acid and sodium fluoride. These three inhibitors reduce gravitropic curvature and cause changes in the phosphorylation of 38- and 50-kDa soluble proteins which show different levels of phosphorylation between lower and upper halves of gravistimulated pulvini. A kinetic analysis of phosphorylation shows that the 38- and 50-kDa soluble proteins exhibit different levels of phosphorylation between lower and upper halves of graviresponsive pulvini at 5 min after initiation of gravistimulation of stems. In addition, the phosphorylation of 63- and 70-kDa proteins from a total membrane preparation increases in lower halves of the pulvini following gravistimulation. These phosphoproteins are not found in the plasma membrane fraction. Taken together, at least four kinds of phosphoproteins are gravi-related. Of these, the 38- and 50-kDa soluble phosphoproteins may be involved in the regulation of early stages of the gravitropic response.     

Effects of glycine on dark-and ligh-induced pulvinar movements and modifications of proton fluxes in the pulvinus of Mimosa pudica during glycine uptake

Glycine (1–50 mM) increases the rate of the dark-induced (scotonastic) movements and decreases the amplitude and the rate of the light-induced (photonastic) movements of the secondary pulvini of Mimosa pudica leaves. The uptake of glycine is accompanied by a long-lasting dose-dependent increase in the alkalinity of the bathing medium of the excised pulvini. The data are in agreement with a H⁺-glycine co-transport mechanism within the pulvinar cells. Fusicoccin (50 M), known to promote H⁺–K⁺ exchange, antagonizes the effects of glycine on the movements and the alkalization of the bathing medium of the excised pulvini. The present results argue for the hypothesis that proton fluxes mediate the scotonastic and photonastic pulvinar movements.Abbreviations Gly glycine - FC fusicoccin - P₁ primary pulvinus - P₂ secondary pulvinus     

Compartmentation of soluble carbohydrates,of starch and of malate in motor organs (pulvini) and other parts of Phaseolus coccineus L. leaves

Quantitative histochemistry was used to investigate the tissue-specific compartmentation of soluble carbohydrates (sucrose, glucose, fructose), starch and malate in the laminar pulvinus, leaf blade and petiole of Phaselous coccineus L. at day and night positions of diurnal leaf movement. Total carbohydrate levels measured in a series of cross sections along individual pulvini of 24-d-old plants showed only small differences between the day and night positions of the respective leaf. In contrast, the level of malate changed during diurnal leaf movement, especially in the central part of a pulvinus. The levels of glucose and fructose in the pulvinus increased towards the transition zones between the pulvinus and lamina, and pulvinus and petiole, and this trend was even more pronounced for starch. By contrast, sucrose levels were highest in the pulvinus proper. The transverse compartmentation of metabolites was studied in distinct, approx. 0.5-mm-thick tissue slices from the central part of a pulvinus. These were dissected further into up to 14 distinct subsamples (bundle, bundle sheath, motor tissues, flanks). Irrespective of the position of the leaf (day or night), the central vascular core and the surrounding bundle sheath had high levels of sucrose (up to 500 mmol-(kg DW)^–1) and low levels of glucose and fructose (below 100 mmol-(kg DW)^–1), while in the cortex the situation was reversed. In the night position the level of sucrose decreased by approx. 30% in the bundle sheath and the central vascular core but not in the other sections. We thus suggest that because of the relatively small diurnal changes in their cortical pools, soluble sugars are not involved in the osmotic processes resulting in leaf movement. In contrast, pulvini from 14-d-old plants showed an interesting diurnal change in starch and malate pools in the outermost layer of the extensor. Here starch increased at night while the malate pool was lowered nearly stoichiometrically. Inverse pool sizes were found in the day position of the respective leaves. Although less significant, the opposite diurnal variation occurred in samples taken from the flexor region. We thus were able to locate areas of different carbohydrate activities in the laminar pulvinus of P. coccineus. The central vascular core, including the bundle sheath, is involved in temporary storage of photoassimilates, and the cortical regions are responsible for osmotically driven leaf movement. The results are discussed with respect to guard-cell physiology.Abbreviations CLP cut-leaf pulvini - ILP intact-leaf pulvini This work was supported by a grant from the Deutsche Forschungsgemeinschaft.